Process for preparing 3aα-H-4α-[3&#39;-propanol]-7aβ-methylhexahydro-1,5-indanedione hemiketal

ABSTRACT

Novel microbial transformation process to selectively convert steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, to 3aα-H-4α-[3&#39;-propanol]-7aβ-methylhexahydro-1,5-indanedione hemiketal having the following structure: ##STR1## This compound can be used as an intermediate to make useful 19-nor steroids.

This is a division of application Ser. No. 768,025, filed Feb. 14, 1977,now U.S. Pat. No. 4,097,335.

BACKGROUND OF THE INVENTION

The transformation of steroids by microorganisms has been widely studiedand documented. Apparently, the earliest such work was by Mamoli andVercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed thereduction of 17-ketosteroids to 17β-hydroxysteroids by fermenting yeast.More recently, Peterson and Murray diclosed the 11α-hydroxylation ofprogesterone with the fungus Rhizopus nigricans; see, U.S. Pat. No.2,602,769 (1952). Also recently, Kraychy et al. in U.S. Pat. No.3,684,657 (1972) discloses the selective microbiological degradation ofsteroidal 17-alkyls by fermenting a steroid containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 toprepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck etal. in U.S. Pat. No. 3,759,791 (1973) disclose the selectivemicrobiological preparation of androst-4-ene-3,17-dione by fermenting asteroid of the cholestane or stigmastane series containing at least 8carbons in the 17-alkyl side chair with Mycobacterium sp. NRRL B-3805.

Also pertinent in the background of the subject invention is the articleby Schubert, K., Bohme, K-H., and Horhold, C. entitled "Formation of lowmolecular degradation products from progesterone by microorganisms,"Steroids 4, 581-586 (1964). These authors described the formation oftricyclic intermediates during the degradation of progesterone byMycobacterium smegmatis.

The culture of the subject invention process, Mycobacterium fortuitumNRRL B-8129 is disclosed in pending applications Ser. Nos. 632,635,filed on Nov. 17, 1975, now U.S. Pat. No. 4,039,381, and 745,113 filedon Nov. 26, 1976, now U.S. Pat. No. 4,062,729. The process conditionsdisclosed in Ser. Nos. 632,635 and 745,113 are not conducive to theoptimal production of compound I of the subject application.

BRIEF SUMMARY OF THE INVENTION

A novel microbial transformation process using mutants which arecharacterized by their ability to selectively degrade steroids with orwithout 17-alkyl side chains of from 2 to 10 carbons atoms, inclusive,and accumulate predominantly compound I in the fermentation beer.

These mutants can be obtained from sterol-degrading microorganisms ofthe following genera by using the mutation procedures disclosed hereinor other mutation procedures Arthrobacter, Bacillus, Brevibacterium,Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, andStreptomyces. A preferred genus is Mycobacterium. Exemplary species ofthis genus are M. phlei, M. Smegmatis, M. rhodochrous, M. mucosum, M.fortuitum, and M. butyricum. Specifically exemplified herein is a mutantmicroorganism, Mycobacterium fortuitum, NRRL B-8129, which is used toselectively degrade steroids with or without 17-alkyl chains containingfrom 2 to 10 carbon atoms, inclusive, to compound I. Examples ofsuitable steroids are sitosterols, cholesterol, stigmasterol,campesterol, and like steroids with 17-alkyl side chains of from 2 to 10carbon atoms, inclusive, and androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, testosterone, andbis-nor acid (3-ketobisnorchol-4-en-22-oic acid). These steroidsubstrates can be in either the pure or crude form.

Also produced in the fermentation beer are lesser amounts of3aα-H-4α-[3'-propanal]-5α-hydroxy-7aβ-methylhexahydro-1-indanonehemiacetal ##STR2## hereinafter referred to as Compound II;

3aα-H-4α[3'-propionicacid]-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone ##STR3##hereinafter referred to as Compound III;

3aα-H-4α-[3'-propanol]-5α-hydroxy-7aβ-methylhexahydro-1-indanone##STR4## hereinafter referred to as Compound IV; and

3aα-H-4α-[3'-propionic acid]-7aβ-methylhexahydro-1,5-indanedione##STR5## hereinafter referred to as Compound V.

DETAILED DESCRIPTION OF THE INVENTION

The Microorganisms

Mutants which are characterized by their ability to selectively degradesteroids with or without 17-alkyl side chains containing from 2 to 10carbon atoms, inclusive, and accumulate predominantly compound I in thefermentation beer, under controlled fermentation conditions, asdisclosed herein, can be obtained by mutating sterol-degradingmicroorganisms of the following genera: Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Mycobacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces. Mycobacterium fortuitum,ATCC 6842, has been mutated, as disclosed herein, to give a laboratorymutant microorganism. The 1974 ATCC Catalogue discloses the followingalongside the listing of ATCC 6842: "J. C. Cruz 2. Cold abscess.Acta-Med. Rio de Janeiro 1:1 (1936). Medium 90 37C." M. fortuitum, ATCC6842, degrades sterols non-selectively to small molecular weightcompounds, e.g., CO₂ +H₂ O. Thus, this microorganism is not suitable asa selective steroid degrader.

Mutation of M. fortuitum, ATCC 6842, using nitrosoguanidine has resultedin the production of a mutant which selectively degrades steroids withor without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive,to produce predominantly compound I in the fermentation beer. Thismutant microorganism of M. fortuitum has been given the accession numberNRRL B-8129, by the Northern Regional Laboratory, U.S. Department ofAgriculture, Peoria, Ill., U.S.A., where it has been deposited in thepermanent collection. A subculture of this microorganism is freelyavailable from this depository by request made thereto. It should beunderstood that the availability of the culture does not constitute alicense to practice the subject invention in derogation of patent rightsgranted with the subject instrument by governmental action.

The morphology and drug sensitivities of M. fortuitum, NRRL B-8129, areindistinguishable from that of the parent M. fortuitum, ATCC 6842. BothM. fortuitum cultures are acid-fast non-motile, non-spore-formingbacilli belonging to the family Mycobacteriaceae of the otherActinomycetales. According to Runyon's classification (Runyon, E. H.1959 Med. Clin. North America 43: 273) it is a nonchromogenic group IVmycobacterium, i.e., it grows rapidly at low temperature to producenonpigmented colonies on relatively simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8129, are clearlydistinguishable in their action on steroid molecules. As disclosedabove, M. fortuitum ATCC 6842 is a degrader of steroids, whereas M.fortuitum NRRL B-8129 is a selective degrader. This property of M.fortuitum NRRL B-8129 makes it highly useful, as disclosed herein.

The mutation of M. fortuitum ATCC 6842 to give M. fortuitum NRRL B-8129was accomplished by the use of nitrosoguanidine. The details of theprocedure are described infra. Though mutation procedures are generallyknown in the art, there is no known art which teaches or even suggeststhe type of mutants, if any, which might be obtained by use of thesubject mutation procedure. Also, though the mutation and transformationprocedures, disclosed herein, are detailed for a Mycobacterium, itshould be understood that similar or equivalent procedures can be usedwith microorganisms of the other genera, as disclosed herein.

The Transformation Process

The selective transformation of the subject invention can be effected ina growing culture of M. fortuitum NRRL B-8129 by either adding theselected steroid substrate to the culture during the incubation period,or incorporating it in the nutrient medium prior to inoculation. Thesteroid can be added singly or in combination with another steroid. Thepreferred, but not limiting, range of concentration of the steroid inthe culture is about 0.1 to about 100 grams per liter. The culture isgrown in a nutrient medium containing a carbon source, for example, anassimilable carbohydrate, and a nitrogen source, for example, anassimilable nitrogen compound or proteinaceous material. Preferredcarbon sources include glucose, brown sugar, sucrose, glycerol, starch,cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogensources include cornsteep liquor, yeast, autolyzed brewer's yeast withmilk solids, soybean meal, cottonseed meal, cornmeal, milk solids,pancreatic digest of casein, fish meal, urea, distillers' solids, animalpeptone liquors, meat and bone scraps, ammonium salts and the like.Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediasince tap water and unpurified ingredients are used as components of themedium. Prior to sterilization of the medium the pH is adjusted to a pHof about 6.5. The pH adjustment can be made with a strong base, forexample, NaOH, KOH, and the like.

A critical feature in the invention process is to maintain the pH atabout 5.5 or lower during the fermentation in order to producepredominantly compound I. Preferably the pH is maintained at about 3.0to about 6.0. This pH maintenance can be done by procedures well knownin the art, for example, by use of CaCo₃ or phosphate buffer in themedium, or by pH control with a base, for example, sodium hydroxide,ammonium hydroxide, and the like, or with an acid, for example, HCl, H₂SO₄, and the like. Further, the pH can be maintained at the desiredlevel by the use of excess amounts of nutritional ingredients themetabolism of which results in a lowered pH. Examples of such compoundsinclude soluble ammonium salts, carbohydrates such as glucose and othermono- and disaccharides, starch and other polysaccharides, and oils andfats such as lard oil and soybean oil.

The transformation process can range from about 72 hours to 15 days. Theincubation temperature during the transformation process can range fromabout 25° C. to about 37° C., with 31° C. being preferred. The contentsof the transformation vessel are aerated with sterilized air andagitated to facilitate growth of the microorganism, and thus, enhancethe effectiveness of the transformation process.

Upon completion of the transformation process, as evidenced by thinlayer chromatography using silica gel plates (E. Merck, Darmstadt) and asolvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane,the desired transformed steroid is recovered by means well known in theart. For example, the fermentation (transformation) reaction mixture,including the fermentation liquor and cells, can be extracted with awater-immiscible organic solvent for steroids. Suitable solvents aremethylene chloride (preferred), chloroform, carbon tetrachloride,ethylene chloride, trichloroethylene, ether, amyl acetate, benzene andthe like.

Alternatively, the fermentation liquor and cells can be first separatedby conventional methods, e.g., filtration or centrifugation, and thenseparately extracted with suitable solvents. The cells can be extractedwith either water-miscible or water-immiscible solvents. Thefermentation liquor, freed of cells, can be extracted withwater-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtratevacuum distilled to dryness. The resulting residue containing thedesired transformed steroid then can be dissolved in 10% chloroform inSkellysolve B and chromatographed on silica gel, using Skellysolve B(isomeric hexanes) and mixtures thereof with increasing amounts of ethylacetate as developing solvent. This procedure elutes the desiredcompound. Upon recrystallization from ethyl acetate, compound I, a 1:1mixture of two epimers, melts at 100°-112° C. A crystalline product canbe obtained by use of a solvent, for example, ethyl acetate. The desiredtransformed steroid can also be obtained from the remaining supernatantupon evaporation of the solvent in the supernatant.

Compound I is useful as an intermediate in the chemical synthesis ofuseful steroids. For example, it can be converted to starting materialfor the process disclosed in U.S. Pat. No. 3,880,884 which discloses aprocess for the total synthesis of useful 19-nor steroids. Thisconversion to starting material can be done by procedures known in theart. See J.A.C.S. 85: 2135-2137.

The following examples are illustrative of the process of the subjectinvention but are not to be construed as limiting. All percentages areby weight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1 Preparation of Mutant M. fortuitum NNRL B-8129 From M.fortuitum ATCC 6842

(a) Nitrosoguanidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the followingsterile seed medium:

    ______________________________________                                        Nutrient Broth (Difco) 8 g/liter                                              Yeast Extract          1 g/liter                                              Sodium Propionate      0.5 g/liter                                            Distilled Water, q.s.  1  liter                                               ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to sterilization at 121°C. for 20 minutes.

The cells are grown to a density of about 5×10⁸ per ml, pelleted bycentrifugation, and then washed with an equal volume of sterile 0.1 Msodium citrate, pH 5.6. Washed cells are resuspended in the same volumeof citrate buffer, a sample removed for titering (cell count), andnitrosoguanidine added to a final concentration of 50 μg/ml. The cellsuspension is incubated at 37° C., in a water bath for 30 minutes, afterwhich a sample is again removed for titering and the remaindercentrifuged down and washed with an equal volume of sterile 0.1 Mpotassium phosphate, pH 7.0. Finally, the cells are resuspended in asterile minimal salts medium, minus a carbon source, consisting of thefollowing:

    ______________________________________                                        NH.sub.4 NO.sub.3    1.0 g/liter                                              K.sub.2 HPO.sub.4    0.25 g/liter                                             MgSO.sub.4 . 7H.sub.2 O                                                                            0.25 g/liter                                             NaCl                 0.005 g/liter                                            FeSO.sub.4 . 7H.sub.2 O                                                                            0.001 g/liter                                            Distilled Water, q.s.                                                                              1  liter                                                 ______________________________________                                    

The pH is adjusted to 7.0 with 1 N HCl prior to sterilization at 121° C.for 20 minutes. The cells are then plated out to select for mutants.

(b) Selection And Isolation Of Mutant M. fortuitum NRRL B-8129

Mutagenized cells, as described above, are diluted and spread ontoplates containing a medium consisting of the following (modified fromFraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295):

    ______________________________________                                        Glycol                10.0 g/liter                                            Na.sub.2 HPO.sub.4    8.4 g/liter                                             KH.sub.2 PO.sub.4     4.5 g/liter                                             NH.sub.4 Cl           2.0 g/liter                                             MgSO.sub.4 . 7H.sub.2 O                                                                             0.3 g/liter                                             FeCl.sub.3 . 6H.sub.2 O                                                                             0.05 g/liter                                            Distilled Water, q.s. 1  liter                                                ______________________________________                                    

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophs produced bythe mutagenesis procedure, e.g., cultures that require vitamins, growthfactors, etc., in order to grow on chemically defined medium areeliminated. After incubation at 28° C. for about 7 days, the resultingcolonies are replicated to test plates suitable for selecting mutantsand then back onto control plates containing the glycerol-based medium.The test plates are prepared as described to Peterson, G. E., H. L.Lewis and J. R. Davis. 1962. "Preparation of uniform dispersions ofcholesterol and other water-insoluble carbon sources in agar media." J.Lipid Research 3: 275-276. The minimal salts medium in these plates isas described above in section (a) of Example 1. Agar (15 g/liter), andan appropriate carbon source (1.0 g/liter), such as sitosterol orandrostenedione (AD), are added and the resulting suspension autoclavedfor 30 minutes at 121° C. The sterile, hot mixture is then poured into asterile blender vessel, blended for several minutes, and then pouredinto sterile Petri plates. Foaming tends to be a problem in thisprocedure but can be reduced by blending when the mixture is hot and byflaming the surface of the molten agar plates. In this manner uniformdispersions of water-insoluble carbon sources are obtained whichfacilitates the preparations of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but not on test platescontaining AD as the sole carbon source, are purified by streaking ontonutrient agar plates. After growth at 28° C., individual clones arepicked from the nutrient agar plates with sterile toothpicks andretested by inoculating gridded plates containing AD as the carbonsource. Purified isolates which still exhibit a phenotype different fromthe parental culture are then evaluated in shake flasks.

(c) Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation mediumconsisting of the following ingredients:

    ______________________________________                                        Glycol                10.0 g/liter                                            Na.sub.2 HPO.sub.4    8.4 g/liter                                             KH.sub.2 PO.sub.4     4.5 g/liter                                             NH.sub.4 Cl           2.0 g/liter                                             MgSO.sub.4 . 7H.sub.2 O                                                                             0.3 g/liter                                             FeCl.sub.3 . 6H.sub.2 O                                                                             0.05 g/liter                                            Distilled Water, q.s. 1  liter                                                ______________________________________                                    

Soyflour (1 g/liter) is blended into the medium and then sitosterol (10g/liter) is also blended into the medium. After the flasks areautoclaved for 20 minutes at 121° C., they are cooled to 28° C. and theninoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. Aloop of cells taken from a slant is used to inoculate a 500-ml flaskcontaining 100 ml of sterile seed medium consisting of the followingingredients:

    ______________________________________                                        Nutrient Broth (Difco) 8 g/liter                                              Yeast Extract          1 g/liter                                              Glycerol               5 g/liter                                              Distilled Water, q.s.  1  liter                                               ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to autoclaving the flasksat 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each500-ml flask containing 100 lm of sterile transformation medium. Theflasks are then incubated at 28° C. to 30° C. on a rotary shaker andsampled at various intervals. Ten ml samples are removed and extractedby shaking with 3 volumes of methylene chloride. Portions of theextracts are analyzed by thin layer chromatography using silica gel andthe solvent system described above, i.e., 2:3 (by volume) ethylacetate-cyclohexane, and by gas-liquid chromatography. Evidence of thepresence of compounds I and V confirms the selective degradation ofsitosterol by the novel mutant produced from the parent M. fortuitumATCC 6842.

EXAMPLE 2 Transformation of Sitosterol To Compound I

The medium used is the same as in Example 1 (c), except that it isadjusted to a pH of about 6.5 with 4 N NaOH. This medium is sterilizedby heating 30 minutes at 121° C., whereupon it is cooled to 28° C. andthen inoculated with 10 parts of a seed culture of the mutant M.fortuitum NRRL B-8129, prepared as described in Example 1 (c). Theinoculated mixture is incubated at 28° C. for 168 hours with agitationto promote submerged growth. As required, the pH is maintained at about5.0 by the addition of NaOH, or an acid, for example, HCl. Progress ofthe biotransformation is followed by chromatography on thin layer silicagel plates in a solvent system consisting of 3:2 (by volume)cyclohexane-ethyl acetate.

Upon completion of the biotransformation the desired product is isolatedby the following procedure. The reaction mixture is extracted withmethylene chloride. This extract is filtered through diatomaceous earthand the filtrate is distilled to dryness. The residue from the filtrateis taken up in 10% chloroform in Skellysolve B and chromatographed onsilica gel, using Skellysolve B and mixtures thereof with increasingamounts of ethyl acetate as developing solvent. This procedure elutesthe desired compound I which has an R_(f) value of 0.37 on thin layerchromatography (cyclohexane-ethyl acetate; 3:2). Compound I melts at100°-112° C.

EXAMPLE 3

By substituting cholesterol for sitosterol in Example 2 there isobtained compound I.

EXAMPLE 4

By substituting stigmasterol in Example 2 for sitosterol there isobtained compound I.

EXAMPLE 5

By substituting campesterol for sitosterol in Example 2 there isobtained compound I.

EXAMPLE 6

By adding a combination of any of the sterols in Examples 2-5, inaddition to sitosterol, or in place of sitosterol, in Example 2 there isobtained compound I.

EXAMPLE 7

By substituting a sterol-degrading microorganism from the generaArthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces, in Example 1 forMycobacterium fortuitum ATCC 6842 there are obtained mutantmicroorganisms which are characterized by their ability to selectivelydegrade steroids with or without a 17-alkyl side chain of from 2 to 10carbon atoms, inclusive, and accumulate predominantly compound I in thefermentation beer.

EXAMPLE 8

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-8129 in Examples 2-6, there is obtained compound I.

EXAMPLE 9

By substituting a sterol-degrading microorganism selected from the groupconsisting of Mycobacterium phlei, M. smegmatis, M. rhodochrous, M.mucosum, and M. butyricum for M. fortuitum ATCC 6842 in Example 1 thereare obtained mutant microorganisms which are characterized by theirability to selectively degrade steroids with or without a 17-alkyl sidechain of from 2 to 10 carbon atoms, inclusive, and accumulatepredominantly compound I in the fermentation beer.

EXAMPLE 10

By substituting the mutants obtained in Example 9 for M. fortuitum NRRLB-8129 in Examples 2-6, there is obtained compound I.

EXAMPLE 11

By substituting a compound selected from the group consisting ofandrost-4-ene-3,17-dione, androsta-1,4-dlene-3, 17-dione,dehydroepiandrosterone, testosterone, and bis-nor acid for sitosterol inExample 2 there is obtained compound I.

EXAMPLE 12

By substituting a combination of two or more compounds selected from thegroup consisting of sitosterol, cholesterol, stigmasterol,androst-4-ene-3-17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, testosterone, and bis-nor acid for sitosterol inExample 2 there is obtained compound I.

EXAMPLE 13

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-8129 in Examples 11 and 12, there is obtained compound I.

EXAMPLE 14

By substituting the mutants obtained in Example 9 for M. fortuitum NRRLB-8129 in Examples 11 and 12, there is obtained compound I.

We claim:
 1. A process for preparing a compound, of the formula ##STR6##which comprises cultivating a mutant microorganism selected from thegroup consisting of Arthrobacter, Bacillus, Brevibacterium,Corynebacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces,said mutant being characterized by its ability to selectively degradesteroids with or without a 17-alkyl side chain of from 2 to 10 carbonatoms, inclusive, and accumulate predominantly3aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indanedione hemiketal inthe fermentation beer, in an aqueous nutrient medium at a pH of 3.0 to6.0 under aerobic conditions in the presence of a steroid with orwithout a 17-alkyl side chain containing from 2 to 10 carbon atoms,inclusive and recovering said compound from the cultivated medium.
 2. Aprocess, according to claim 1, wherein said mutant microorganism iscultivated in an aqueous nutrient medium under aerobic conditions in thepresence of a mixture of two or more steroids.
 3. A process, accordingto claim 1, wherein said steroid is selected from the group consistingof sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, testosterone, and bis-nor acid.
 4. A process,according to claim 2, wherein said steroid mixture is selected from thegroup consisting of sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, testosterone, and bis-nor acid.
 5. A process forpreparing a fermentation beer containing predominantly the compound ofthe formula ##STR7## which comprises cultivating a mutant microorganismselected from the group consisting of Arthobacter, Bacillus,Brevibacterium, Corynebacterium, Nocardia, Protaminobacter, Serratia,and Streptomyces, said mutant being chracterized by its ability toselectively degrade steroids with or without a 17-alkyl side chain offrom 2 to 10 carbon atoms, inclusive, and accumulate predominantly3-aα-H-4α-[3'-propanol]-7aβ-methylhexahydrol-1,5-indanedione hemiketulin the fermentation beer, in an aqueous nutrient medium at a pH of 3.0to 6.0 under aerobic conditions in the presence of a steroid with orwithout a 17-alkyl side chain containing from 2 to 10 carbon atoms,inclusive.
 6. A process, according to claim 5, wherein said mutantmicroorganism is cultivated in an aqueous nutrient medium under aerobicconditions in the presence of a mixture of two or more steroids.
 7. Aprocess, according to claim 5, wherein said steroid is selected from thegroup consisting of sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, testosterone, and bis-nor acid.
 8. A process,according to claim 6, wherein said steroid mixture is selected from thegroup consisting of sitosterol, cholesterol, stigmasterol, campesterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, testosterone, and bis-nor acid.